Claudia A. Mueller

Serum protein profiling by SELDI mass spectrometry: detection of multiple variants of serum amyloid alpha in renal cancer patients

The molecular analysis of serum is an important field for the definition of potential diagnostic markers or disease-related protein alterations. Novel proteomic technologies such as the mass spectrometric-based surface-enhanced laser desorption/ionization (SELDI) ProteinChip® technique facilitate a rapid and reproducible analysis of such protein mixtures and affords the researcher a new dimension in the search for biomarkers of disease. Here, we have applied this technology to the study of a cohort of serum samples from well-characterized renal cell carcinoma patients for the identification of such proteins by comparison to healthy controls. We detected and characterized haptoglobin 1 α and serum amyloid α-1 (SAA-1) as disease related, in addition to an as-yet-unidentified marker of 10.84 kDa. Of particular note is the detection of multiple variants of SAA-1 in multiplex that have not been described in the sera of cancer patients. SAA-1 is detected as full-length protein, des-Arginine and des-Arginine/des-Serine variants at the N terminus by SELDI. In addition, we could also detect a low-abundant variant minus the first five N-terminal amino acids. Such variants may impact the function of the protein. We conclude the technique to be a reproducible, fast and simple mode for the discovery and analysis of marker proteins of disease in serum.

Cathepsin D is present in human eccrine sweat and involved in the postsecretory processing of the antimicrobial peptide DCD-1L

The protein pattern of healthy human eccrine sweat was investigated and 10 major proteins were detected from which apolipoprotein D, lipophilin B, and cathepsin D (CatD) were identified for the first time in human eccrine sweat. We focused our studies on the function of the aspartate protease CatD in sweat. In vitro digestion experiments using a specific fluorescent CatD substrate showed that CatD is enzymatically active in human sweat. To identify potential substrates of CatD in human eccrine sweat LL-37 and DCD-1L, two antimicrobial peptides present in sweat, were digested in vitro with purified CatD. LL-37 was not significantly digested by CatD, whereas DCD-1L was cleaved between Leu44 and Asp45 and between Leu29 and Glu30 almost completely. The DCD-1L-derived peptides generated in vitro by CatD were also found in vivo in human sweat as determined by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. Furthermore, besides the CatD-processed peptides we identified additionally DCD-1L-derived peptides that are generated upon cleavage with a 1,10-phenanthroline-sensitive carboxypeptidase and an endoprotease. Taken together, proteolytic processing generates 12 DCD-1L-derived peptides. To elucidate the functional significance of postsecretory processing the antimicrobial activity of three CatD-processed DCD-1L peptides was tested. Whereas two of these peptides showed no activity against Gram-positive and Gram-negative bacteria, one DCD-1L-derived peptide showed an even higher activity against Escherichia coli than DCD-1L. Functional analysis indicated that proteolytic processing of DCD-1L by CatD in human sweat modulates the innate immune defense of human skin.

Detection of dermcidin-derived peptides in sweat by ProteinChip® Technology

Recently, a novel antimicrobial peptide DCD-1, derived from the Dermcidin (DCD) gene and secreted by sweat glands, has been described by Schittek et al. [Nat. Immunol. 2 (2001) 1133.]. Here we describe the application of the surface-enhanced laser desorption/ionisation (SELDI) technology for the detection of DCD-1 and other dermcidin-derived peptides directly from microlitre amounts of human sweat. The advantages of the technique are as follows: (a) it can be carried out with ease and rapidity; (b) multiple samples can be processed simultaneously; (c) prior purification is not required; and (d) only a limited sample volume is necessary for both protein profiling and semiquantitation. Profiling of human sweat from various donors revealed that in addition to DCD-1, other DCD-derived peptide species were also present in significant quantities. Four of five identified peptides were DCD-1 related, while the fifth corresponded to a portion of the DCD protein outside the DCD-1 core. This provides clues as to how the novel protein is processed to its active form, though further work remains to elucidate this fully. Thus, we have demonstrated the applicability of such technology to the detection of DCD-1 and for the protein profiling of sweat in general. Such studies could reveal valuable new biomarkers for diagnosis and treatment of skin and sweat gland disorders.

Secretion of ERP57 is important for extracellular matrix accumulation and progression of renal fibrosis, and is an early sign of disease onset.

Summary Renal fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), which compromises organ function by replacing normal organ tissue. The molecular mechanisms leading to renal fibrosis are not fully understood. Here we demonstrated that TGF&bgr;1, AGT or PDGF stimulation of renal cells resulted in endoplasmic reticulum (ER) stress followed by activation of the protective unfolded protein response pathway and a high secretory level of protein disulfide isomerase ERP57 (also known as PDIA3). The TGF&bgr;1-induced impairment of ER function could be reversed by treatment with BMP7, suggesting a specific involvement in renal fibrosis. A clear correlation between the degree of fibrosis, ER stress and the level of ERP57 could be seen in fibrosis animal models and in biopsies of renal fibrosis patients. Protein interaction studies revealed that secreted ERP57 exhibits a strong interaction with ECM proteins. Knockdown of ERP57 or antibody-targeted inhibition of the secreted form significantly impaired the secretion and accumulation of ECM. Moreover, ERP57 was excreted in the early stages of chronic kidney disease, and its level in urine correlated with the degree of renal fibrosis, suggesting that the secretion of ERP57 represents one of the first signs of renal fibrosis onset and progression.

Proteomics characterization of cell model with renal fibrosis phenotype: osmotic stress as fibrosis triggering factor.

Renal fibroblasts are thought to play a major role in the development of renal fibrosis (RF). The mechanisms leading to this renal alteration remain poorly understood. We performed differential proteomic analyses with two established fibroblast cell lines with RF phenotype to identify new molecular pathways associated with RF. Differential 2-DE combined with mass spectrometry analysis revealed the alteration of more than 30 proteins in fibrotic kidney fibroblasts (TK188) compared to normal kidney fibroblast (TK173). Among these proteins, markers of the endoplasmic reticulum (ER) stress- and the unfolded protein response (UPR) pathway (GRP78, GRP94, ERP57, ERP72, and CALR) and the oxidative stress pathway proteins (PRDX1, PRDX2, PRDX6, HSP70, HYOU1) were highly up-regulated in fibrotic cells. Activation of these stress pathways through long time exposition of TK173, to high NaCl or glucose concentrations resulted in TK188 like phenotype. Parallel to an increase in reactive oxygen species, the stressed cells showed significant alteration of fibrosis markers, ER-stress and oxidative stress proteins. Similar effects of osmotic stress could be also observed on renal proximal tubule cells. Our data suggest an important role of the ER-stress proteins in fibrosis and highlights the pro-fibrotic effect of osmotic stress through activation of oxidative stress and ER-stress pathways.

Expression of cadherin-8 in renal cell carcinoma and fetal kidney.

Cadherins represent a family of calcium‐dependent cell adhesion molecules with an important regulatory function for maintenance of tissue architecture. Alterations of cadherin expression have been demonstrated in the development and progression of different epithelial tumors. In renal cell carcinoma (RCC), the majority of tumors express N‐cadherin and cadherin‐6. Screening a series of 16 RCC cell lines for the expression of different novel type II cadherins by RT‐PCR revealed a complex pattern of cadherin expression: cadherins 6 and 14 were expressed in most of the RCC cell lines, whereas cadherins 11, 12 and 13 could not be detected at all. Interestingly, cadherin‐8, previously shown in mice to be restricted to the CNS and thymus during development, was detected by RT‐PCR, immunofluorescence and in situ hybridization in 4 of 16 RCC cell lines as well as in paraffin sections of the corresponding human RCC biopsies. In normal renal tissue, however, cadherin‐8 could be detected only during the early stages of kidney development. These results suggest that alterations of type II cadherin expression may play a role in RCC development. In particular, cadherin‐8 may be involved in both kidney morphogenesis as well as tumorigenesis in some types of RCC.

Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate.

Methotrexate was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders; its molecular mechanism remains elusive. To understand the molecular mechanism by which methotrexate induces apoptosis, we analyzed the resulting intracellular protein changes in methotrexate-treated acute promyelocytic leukaemia (HL-60) cells by cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry. Initial CL-DIGE analysis revealed that 24 proteins were differentially expressed (p<0.05) in the HL-60 cell proteome after treatment with 2.5microM methotrexate for 72h. We found that three structural alpha4, alpha5, alpha7 proteasome subunits, a non-catalytic beta3 and two 26S regulatory proteasome subunits were down-regulated in methotrexate-treated HL-60 cells. Western blot analyses further showed that the inhibition of proteasome subunits is accompanied by suppression of NF-kappaB subunits and promotes the accumulation of ubiquitinated proteins. Furthermore, methotrexate activated unfolded protein response by inducing the expression of endoplasmic reticulum-resident proteins such as calreticulin, protein disulphide isomerase A3 and A4, and 78kDa glucose regulated protein in a time-dependent manner. Altogether, our findings demonstrated that targeting NF-kappaB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells.

Haptoglobin-α1, -α2, vitamin D-binding protein and apolipoprotein C-III as predictors of etanercept drug response in rheumatoid arthritis

IntroductionThe introduction of tumor necrosis factor-alpha (TNF-α) antagonists has substantially improved patient’s clinical outcome in rheumatoid arthritis (RA). However, nearly 20% to 40% of RA patients do not respond to anti-TNF-α treatment strategies. To identify valid predictors of TNF-α antagonist response in RA, serum proteome profiles from responders (R) and non-responders (NR) to etanercept, a soluble recombinant TNF-α receptor/IgG Fc fusion protein receptor, were compared in a prospective cohort study.MethodsIn this clinical study 50 RA patients with inadequate response to conventional DMARDs were included and treated with etanercept. The primary efficacy endpoint was response according to the European League against Rheumatism (EULAR) improvement criteria. Serum samples collected prior to initiation and after six months of etanercept therapy were cleared of the most abundant major proteins by immunoaffinity chromatography. After separation by two-dimensional differential gel electrophoresis (2D-DIGE) and identification by mass spectrometry (MS) data were validated by Western blot analysis.ResultsAfter six months of etanercept treatment 62% (n = 31) of RA patients achieved response. Haptoglobin-α1 (Hp-α1) and -α2 (Hp-α2) and vitamin D-binding protein (VDBP) were found to be significantly upregulated in responder sera (P ≤0.02) at study entry. In contrast, apolipoprotein C-III (ApoC-III) showed significantly higher levels in non-responders (P = 0.0162). At study end ApoA-II, Hp-α1, Hp-α2 and VDBP were identified to be expressed at significantly higher levels (P <0.05) in responder sera.ConclusionsBy application of clinical proteomics in immunodepleted sera we could identify and validate for the first time Hp-α1, -α2, VDBP and ApoC-III as potential biomarkers for prediction of etanercept drug response in RA.

Correction: Secretion of ERP57 is important for extracellular matrix accumulation and progression of renal fibrosis, and is an early sign of disease onset (doi:10.1242/jcs.125088)

There was an error published in J. Cell Sci. (2013) 126, [3649–3663][1] ([doi:10.1242/jcs.125088][2]). The affiliations for Marwa Eltoweissy were incorrect. The correct affiliations are as given below. Hassan Dihazi1, Gry Helene Dihazi1, Asima Bibi1, Marwa Eltoweissy1,2, Claudia A. Mueller3,

Impaired immune defense in hemodialysis patients: Role of α-defensins?

The mechanisms underlying the impaired immune response in hemodialysis (HD) patients are not completely understood. The α‐defensins human neutrophil peptides‐1, 2, and 3 are low molecular weight peptides with antimicrobial activity and important effector molecules of innate immune responses. We now examined the expression of these peptides in HD patients. Seventy‐six patients on chronic HD treatment (mean time on HD 5.8 years; mean age 70 years) were studied and compared with 38 healthy volunteers and 20 patients with infections and normal renal function. Expression of α‐defensins was analyzed semiquantitatively in leukocytes on the messenger RNA (mRNA) level by reverse transcriptase polymerase chain reaction; the α‐defensin protein levels in serum were detected by enzyme‐linked immunosorbent assay. α‐Defensin concentrations (140 ± 10.5 ng/mL; mean ± standard error of the mean) as well as mRNA levels in leukocytes (82.9 ± 7.9 arbitrary units [a.u.]) in HD patients were not significantly different from those in healthy volunteers (156 ± 15.2 ng/mL; 81.4 ± 11.3 a.u.). Defensin levels were independent of the time of the patient on HD and their age. During infection periods (mean increase of the C‐reactive protein to 161 ± 17.3 mg/L), defensin serum levels increased to 321 ± 65 ng/mL (P < 0.005) and mRNA expression in leukocytes to 159 ± 19.2 a.u. (P < 0.05). These increases were not significantly different from those in patients with normal renal function (298 ± 46.8 ng/mL and 128 ± 9.1 a.u., respectively) suffering from infections (C‐reactive protein 222 ± 26.6 mg/L). Our results suggest that the impaired immune defense in dialysis patients is not due to a deficiency in α‐defensins in these patients as neither basal levels nor expression during infections were reduced compared with subjects with normal renal function.